Coumarin and rhodamine dyes are known as bright fluorescent labels with large absorption coefficients, high fluorescent quantum yields, and low degree of triplet formation. They are widely used both as laser dyes and fluorescent compounds for labeling proteins, nucleic acids, lipids, carbohydrates, toxins, hormones and other biomolecules (for examples, see: R. P. Haugland, A Guide to Fluorescent Probes and Labeling Technologies, Invitrogen, Carlsbad, 2005, pp. 11-37, 77-78).
Rhodamines are generally more photostable than coumarins. Therefore they are often applied for practical implementations of new physical concepts that require high light intensities and/or the detection of the single photostable and fluorescent molecules. For example, it was demonstrated that the diffraction limit of an optical microscope can be overcome by switching between the dark and bright states of a fluorescent marker (S. W. Hell, in Single Molecule Spectroscopy in Chemistry, Physics and Biology, Eds: A. Gräslund, R. Rigler, J. Widengren, Springer, Berlin, 2010, pp. 365-398). In particular, a very important novel method, the stimulated emission depletion (STED) microscopy, uses the ground (singlet) state of the fluorophore (S0) as a dark state, and the first excited state (S1) as a bright one. In practical applications of the STED method, a focused pulse excites fluorescence in a small spot (with dimensions limited by diffraction), and then a red-shifted doughnut-shaped STED beam switches off the fluorescence of the excited molecules by stimulated emission (S1→S0) everywhere, except in the very center of the doughnut, where the quenching intensity is zero. For squeezing the fluorescence to a very small central spot, the depletion rate should exceed the rate of the spontaneous transition to the ground state S0. Fluorescent lifetimes of organic fluorophores (τfl˜10−9 s) and their optical cross-sections of the S1→S0 transitions (σ˜10−16 cm2) imply that the STED-pulse should have a very high power ISTED>>IS≡(στfl)−1˜1025 photons/(cm2×s)≅10 MW/cm2. (IS is a threshold intensity depending on the dye employed and the depletion wavelength used.) The resolution enhancement scales roughly with √{square root over (1+ISTED/IS)}.
These huge light intensities inevitably cause photobleaching of fluorophores, and therefore STED microscopy ultimately requires the most photostable fluorescent dyes. The far-field optical “nanoscopy” based on the STED principle provides an optical resolution of 30-60 nm with organic fluorophores.
Along with the relatively long lifetimes of the excited states (e.g. 1-20 ns), other important qualities of the STED and common fluorescent dyes are high fluorescent quantum yields (Φfl) and oscillator strengths (high absorption coefficients, ε), low rate of triplet state formation, sufficient solubility in water or aqueous buffers and a reactive group (with a linker) for attaching the dye to a biological object or any other structure of interest. High Φfl-values of the fluorescent labels conjugated with biomolecules are very important, as they improve the sensitivity of the imaging method. Moreover, if a resolution on the molecular scale is desired, or if only single molecules remain in the effective detection volume, the fluorescent dyes should be suitable for single molecule detection (e.g. in the method of fluorescence correlation spectroscopy—FCS).
Water is the preferred solvent for operating with the reactive fluorescent dyes, because the conjugation reactions involving biologically relevant macromolecules (proteins, nucleic acids, carbohydrates, etc.) need to be performed in water or aqueous buffers. A marker is usually dissolved in an organic solvent, such as DMF or DMSO, and then added to the aqueous solution of the substrate. High concentrations of an organic solvent may cause protein denaturizing, and hence should be avoided. On the other hand, a low coupling efficiency may be observed if the amount of the organic solvent is too low and the marker precipitates in the reaction mixture. Water-soluble fluorescent markers are advantageous in this regard, because they do not require any organic solvents at all. Moreover, hydrophilic labels are less prone to aggregation and to non-specific binding with biological objects, especially membranes.
Biological applications require fluorescent dyes absorbing and emitting in the red spectral region, because the excitation in this area reduces the background originating from autofluorescence of the cells (evident with UV and blue excitation). Very convenient is the excitation by the red He—Ne laser at 633 nm or with the 635 nm spectral line of a red diode laser, as well as with the 647 nm line of the krypton ion laser or with a diode laser emitting at 650 nm. For two-color applications with coumarin and rhodamine dyes, diode lasers emitting at 405 nm or 488 nm, respectively, constitute other convenient excitation sources.
Another important feature of a fluorescent dye is the Stokes shift (distance between the absorption and emission maxima measured in nm or cm−1). Fluorescent dyes with large Stokes shifts can be used alone or together with emitters possessing small Stokes shifts in various imaging techniques. For example, a pair of dyes emitting approximately at the same wavelength with well separated absorption bands may be used for labeling, detection and colocalization of two different (biological) targets. A great advantage of this approach is that only one detection channel is used. The “cross-talk” observed in the course of the excitation with two different light sources (lasers) has to be low. Photostable and brightly fluorescent dyes with large Stokes shifts are rare and only few of them are commercially available. Many of these dyes contain a coumarin fragment as the fluorophore. For example, the “Mega Stokes” dyes from Dyomics are coumarins absorbing at about 500-520 nm, and emitting in the region of 590-670 nm [P. Czerney, M. Wenzel, B. Schwender, F. Lehmann, EP 1318177 B1, 5 May 2002; U.S. Pat. No. 7,563,907 B2, 21 Jul. 2009]. Another practically useful coumarin dye is Alexa Fluor™ 430 with an absorption maximum at 434 nm and an emission band at 539 nm (Invitrogen). However, the chemical structures of many commercially available fluorescent dyes with large Stokes shifts are unknown. For example the structures of Pacific Orange™ (abs. 390 nm, emission 540 nm; Invitrogen) and V500 (abs. 410 nm, emission 500 nm; BD Horizon™) have not been disclosed. The common features of coumarin dyes are large Stokes shifts, as well as moderate photostability and relatively low fluorescence quantum yields in polar solvents.
Analysis of the disclosed structures of the commercial fluorescent dyes matching the excitation with a red He—Ne laser (633 nm) or the 635 nm spectral line of red diode lasers reveals that there is only one rhodamine among them: Alexa 633 was reported to be a “sulfonated rhodamine derivative” [J. E. Berlier et al., J. Histochem. Cytochem. 2003, 51, 1699-1712], and only in 2007 its structure has been reported [B. Agnew, K. R. Gee, T. G. Nyberg (Invitrogen), US Pat. 2007/0249014]. Up to now, the highest values for the adsorption and emission maxima have been achieved for rhodamines with a rigidized xanthene fragment obtained from tetrafluoro- or tetrachlorophthalic anhydrides. For example, absorption and emission maxima of 630 and 655 nm, respectively, have been observed in 8 M urea solution [L. G. Lee, R. J. Graham, W. E. Werner, E. Swartzman, L. Lu, (Apptera Corp., USA), U.S. Pat. No. 6,372,907 (16 Apr. 2002)]. The disadvantage of the fluorescent dye with four fluorine atoms disclosed in the document cited above is its high lipophilicity (low polarity) and therefore low solubility in water or aqueous buffers. Another drawback of this compound (as well as tetrachloro rhodamine JA 407 [compound 40 in WO 2005/003086] is that they have a free carboxylic group which may give a colorless and non-fluorescent cyclic ester form. Similar spectral values (624 and 644 nm for the absorption and emission, respectively) have been recorded in ethanol for ethyl esters of the tetrachloro rhodamines JA 407-E and AZ 84-AZ 95 [compounds 41 and 71-82 in WO 2005/003086].
Though rhodamine dyes disclosed in U.S. Pat. No. 6,372,907 and in WO 2005/003086 represent valuable intermediates, they lack any suitable reactive site for an attachment to biomolecules. The carboxylic group (COOH) on the benzene ring in the ortho position to the xanthene fragment in the compounds described in U.S. Pat. No. 6,372,907, as well as the COOH-group in the acid JA 407 [compound 40 in WO 2005/003086] are sterically hindered and therefore, less reactive. Moreover, the reaction of this carboxylic acid with primary amino groups (e.g. in various biomolecules, like peptides, proteins, lipids, etc.) would give amides which are known to form colorless and non-fluorescent cyclic spiroamides (due to addition of the NH-group across the tetrasubstituted C9═C8a/8b double bond in the central xanthene ring).
Some drawbacks of the red-emitting rhodamines mentioned above were overcome in EP 2253635 (WO2010124833) in which new photostable, bright, amino and thiol reactive rhodamines for labeling and imaging have been disclosed. However, the fluorescence quantum yield decreased considerably after conjugation with antibodies (from 78-80% to 40-48%, depending on the nature of the protein), and the stability of the active esters was found to be rather low.
The alteration in the fluorescence quantum yield after bioconjugation (compared to that of a free dye in an aqueous buffer solution) is a very important issue. In the course of the labeling procedure, a fluorescent dye is bound to a protein (e.g. streptavidin, primary or secondary antibodies, etc.), and the fluorescence signal of the bioconjugate (which is very often further diluted in the course of the immunolabeling procedure) is observed in a light microscope.
As a rule, the high fluorescence quantum yield of a (hydrophilic) fluorescent dye decreases upon biocojugation [a) V. P. Boyarskiy, V. N. Belov, R. Medda, B. Hein, M. Bossi, S. W. Hell, Chem. Eur. J. 2008, 14, 1784-1792; b) K. Kolmakov, V. N. Belov, J. Bierwagen, C. Ringemann, V. Müller, C. Eggeling, S. W. Hell, Chem. Eur. J. 2010, 16, 158-166]. For obvious reasons, there is a great demand of fluorescent dyes with a minimal decrease in the fluorescence quantum yield caused by bioconjugation.
In view of the drawbacks of the fluorescent dyes of the prior art, the main object of the present invention was to provide a novel general approach to hydrophilic fluorescent dyes with improved properties, namely photostability in aqueous solutions, solubility in pure water and aqueous buffers, high values for fluorescence quantum yield in the free state and after conjugation with proteins, which would be suitable for microscopy applications with very high light intensities such as STED and FCS.
This objective has been achieved by providing novel fluorescent dyes with phosphorylated hydroxymethyl groups according to claims 1-3, a method for preparing such dyes which are phosphorylated coumarins and rhodamines according to claims 4 and 5, precursor compounds for the synthesis of said coumarins and rhodamines according to claims 6 and 7, as well as the uses of the disclosed novel fluorescent dyes according to claims 8-11.